The present invention relates to intracellular signaling molecules, in particular the Nod2 protein and nucleic acids encoding the Nod2 protein. The present invention provides assays for the detection of Nod2 and Nod2 polymorphisms associated with disease states. The present invention further provides inhibitors of Nod2 signaling and methods for identifying Nod2 pathway components.
Inflammatory bowel diseases (IBD) are defined by chronic, relapsing intestinal inflammation of obscure origin. IBD refers to two distinct disorders, Crohn""s disease and ulcerative colitis (UC). Both diseases appear to involve either a dysregulated immune response to GI tract antigens, a mucosal barrier breach, and/or an adverse inflammatory reaction to a persistent intestinal infection. The GI tract luminal contents and bacteria constantly stimulate the mucosal immune system, and a delicate balance of proinflammatory and anti-inflammatory cells and molecules maintains the integrity of the GI tract, without eliciting severe and damaging inflammation. It is unknown how the IBD inflammatory cascade begins, but constant GI antigen-dependent stimulation of the mucosal and systemic immune systems perpetuates the inflammatory cascade and drives lesion formation.
There is no known cure for IBD, which afflicts 2 million Americans. Current methods of managing IBD symptoms cost an estimated $1.2 billion annually in the United States alone.
In patients with IBD, ulcers and inflammation of the inner lining of the intestines lead to symptoms of abdominal pain, diarrhea, and rectal bleeding. Ulcerative colitis occurs in the large intestine, while in Crohn""s, the disease can involve the entire GI tract as well as the small and large intestines. For most patients, IBD is a chronic condition with symptoms lasting for months to years. It is most common in young adults, but can occur at any age. It is found worldwide, but is most common in industrialized countries such as the United States, England, and northern Europe. It is especially common in people of Jewish descent and has racial differences in incidence as well. The clinical symptoms of IBD are intermittent rectal bleeding, crampy abdominal pain, weight loss and diarrhea. Diagnosis of IBD is based on the clinical symptoms, the use of a barium enema, but direct visualization (sigmoidoscopy or colonoscopy) is the most accurate test. Protracted IBD is a risk factor for colon cancer. The risk for cancer begins to rise significantly after eight to ten years of IBD.
Some patients with UC only have disease in the rectum (proctitis). Others with UC have disease limited to the rectum and the adjacent left colon (proctosigmoiditis). Yet others have UC of the entire colon (universal IBD). Symptoms of UC are generally more severe with more extensive disease (larger portion of the colon involved with disease).
The prognosis for patients with disease limited to the rectum (proctitis) or UC limited to the end of the left colon (proctosigmoiditis) is better then that of full colon UC. Brief periodic treatments using oral medications or enemas may be sufficient. In those with more extensive disease, blood loss from the inflamed intestines can lead to anemia, and may require treatment with iron supplements or even blood transfusions. Rarely, the colon can acutely dilate to a large size when the inflammation becomes very severe. This condition is called toxic megacolon. Patients with toxic megacolon are extremely ill with fever, abdominal pain and distention, dehydration, and malnutrition. Unless the patient improves rapidly with medication, surgery is usually necessary to prevent colon rupture.
Crohn""s disease can occur in all regions of the gastrointestinal tract. With this disease intestinal obstruction due to inflammation and fibrosis occurs in a large number of patients. Granulomas and fistula formation are frequent complications of Crohn""s disease. Disease progression consequences include intravenous feeding, surgery and colostomy.
The most commonly used medications to treat IBD are anti-inflammatory drugs such as the salicylates. The salicylate preparations have been effective in treating mild to moderate disease. They can also decrease the frequency of disease flares when the medications are taken on a prolonged basis. Examples of salicylates include sulfasalazine, olsalazine, and mesalamine. All of these medications are given orally in high doses for maximal therapeutic benefit. These medicines are not without side effects. Azulfidine can cause upset stomach when taken in high doses, and rare cases of mild kidney inflammation have been reported with some salicylate preparations.
Corticosteroids are more potent and faster-acting than salicylates in the treatment of IBD, but potentially serious side effects limit the use of corticosteroids to patients with more severe disease. Side effects of corticosteroids usually occur with long term use. They include thinning of the bone and skin, infections, diabetes, muscle wasting, rounding of faces, psychiatric disturbances, and, on rare occasions, destruction of hip joints.
In IBD patients that do not respond to salicylates or corticosteroids, medications that suppress the immune system are used. Examples of immunosuppressants include azathioprine and 6-mercaptopurine. Immunosuppressants used in this situation help to control IBD and allow gradual reduction or elimination of corticosteroids. However, immunosuppressants cause increased risk of infection, renal insufficiency, and the need for hospitalization.
Clearly there is a great need for identification of the molecular basis of IBD, or its associated disorders Crohn""s disease and ulcerative colitis.
The present invention relates to intracellular signaling molecules, in particular the Nod2 protein and nucleic acids encoding the Nod2 protein. The present invention provides assays for the detection of Nod2 and Nod2 polymorphisms associated with disease states. The present invention further provides inhibitors of Nod2 signaling and methods for identifying Nod2 pathway components.
Thus, in some embodiments, the present invention provides an isolated and purified nucleic acid comprising a sequence encoding a protein selected from the group consisting of SEQ ID NOs: 2,3 and 34. In some embodiments, the nucleic acid sequence is operably linked to a heterologous promoter. In some embodiments, the nucleic acid sequence is contained within a vector. In some further embodiments, the vector is within a host cell.
In other embodiments, the present invention provides an isolated and purified nucleic acid sequence that hybridizes under conditions of low stringency to a nucleic acid selected from the group consisting of SEQ ID NO:1 and 33. In some embodiments, the nucleic acid sequence encodes a protein that activates NF-xcexaB. In other embodiments, the present invention provides a vector comprising the nucleic acid sequence. In still other embodiments, the vector is within a host cell. In some embodiments, the host cell is located in an organism selected from the group consisting of a plant and an animal.
In yet other embodiments the present invention provides a protein encoded by a nucleic acid selected from the group consisting of SEQ ID NOs:1 and 33 and variants thereof that are at least 80% identical to SEQ ID NOs: 1 and 33, wherein the protein has at least one activity of Nod2. In some embodiments, the activity is activation of NF-xcexaB. In other embodiments, the activity is binding to RICK. In some embodiments, the protein is at least 90% identical to SEQ ID NOs:1 and 33. in other embodiments, the protein is at least 95% identical to SEQ ID NOs:1 and 33.
In still further embodiments, the present invention provides a method for producing variants of Nod2 comprising: providing a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1 and 33; mutagenizing the nucleic acid sequence; and screening the variant for Nod2 activity.
In additional embodiments, the present invention provides a nucleic acid encoding Nod2, wherein the Nod2 competes for binding to NF-xcexaB with a protein encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1 and 33.
In other embodiments, the present invention provides a composition comprising a nucleic acid that inhibits the binding of at least a portion of a nucleic acid selected from the group consisting of SEQ ID NOs:1 and 33 to their complementary sequences. In yet other embodiments, the present invention provides a polynucleotide sequence comprising at least fifteen nucleotides capable of hybridizing under stringent conditions to the isolated nucleotide sequence selected from the group consisting of SEQ ID NOs:1 and 33.
The present invention also provides a method for detection of a polynucleotide encoding Nod2 protein in a biological sample suspected of containing a polynucleotide encoding Nod2. The method includes hybridizing the polynucleotide sequence selected from the group consisting of SEQ ID NOs:1 and 33 and variants thereof that are at least 80% identical to SEQ ID NOs: 1 and 33 (and wherein the protein has at least one activity of Nod2) to the nucleic acid of the biological sample to produce a hybridization complex. In some embodiments, the method further includes the step of detecting the hybridization complex, wherein the presence of the hybridization complex indicates the presence of a polynucleotide encoding Nod2 in the biological sample. In some embodiments, prior to hybridization, the nucleic acid of the biological sample is amplified.
The present invention further provides a method for screening compounds for the ability to alter Nod2 activity, comprising: providing: a first polypeptide sequence comprising at least a portion of Nod2; ii) a second polypeptide sequence comprising at least a portion of a protein known to interact with Nod2; and iii) one or more test compounds; combining in any order, the first polypeptide sequence comprising at least a portion of Nod2, the second polypeptide sequence comprising at least a portion of a protein known to interact with Nod2, and one or more test compounds under conditions such that the first polypeptide sequence, the second polypeptide sequence, and the test compound interact; and detecting the presence or absence of an interaction between the polypeptide sequence comprising at least a portion of Nod2 and the polypeptide sequence comprising at least a portion of a protein known to interact with Nod2. In some embodiments, the first polypeptide sequence is selected from the group consisting of SEQ ID NOs: 2-17 and 34. In some embodiments, the second polypeptide comprises RICK.
The present invention also provides a method of identifying individuals suffering from Crohn""s disease or at risk of developing Crohn""s disease comprising: providing nucleic acid from a patient; wherein the nucleic acid comprises a Nod2 allele; and detecting a mutation in the nucleic acid, wherein the mutation results in increased NF-xcexaB activation. In some embodiments, the mutation is in said Nod2 allele. In some embodiments, the mutation is a cytosine residue insertion. In still further embodiments, the mutation causes a deletion of at least one LRR repeat of Nod2. In some embodiments, the detecting step is accomplished by hybridization analysis. In some embodiments, the method further includes the step of providing a prognosis to the patient based on the presence or absence of the mutation.
In yet other embodiments, the present invention provides a kit for determining if a subject is at risk of developing Crohn""s disease comprising: at least one reagent that specifically detects a mutation in a Nod2 allele; and instructions for determining that the subject is at increased risk of developing Crohn""s disease.
The present invention also provides a purified polypeptide selected from the group consisting of SEQ ID NOs:2, 3, and 34.
The present invention additionally provides a compound capable of inhibiting the binding of a Nod2 to a RICK polypeptide.